Purpose: Transforming growth factor beta-induced protein (TGFBIp) is a widely expressed extracellular matrix protein that plays roles in cell adhesion and migration, differentiation, apoptosis, bone morphogenesis, and carcinogenesis. Mutations of TGFBIp have been linked to stromal corneal dystrophies, a group of protein conformational diseases characterized by abnormal protein aggregations in the cornea. However, the underlying pathogenic mechanism remains elusive due to a lack of insight into the molecular properties of the disease-causing mutants. In the current study, we applied spectroscopic tools to compare the conformation and protein stability of recombinant wild-type (WT) TGFBIp to two dystrophic mutants, R124C and R555W. Methods: A serum-free expression system was used to produce the recombinant TGFBIp proteins. Fluorescence and far-ultraviolet circular dichroism spectroscopies were used to compare WT and dystrophic mutants under various conditions. Results: Our results showed that dystrophic mutants were processed differentially by the expressing cells and produced different proteolytic fragment patterns by proteolysis. Intrinsic tryptophan fluorescence studies revealed moderate shifts in the emission maxima and increased quenching by iodide ion of mutant TGFBIp, suggesting a different conformation than WT protein. Denaturation experiments indicated a difference in protein stability between WT and mutant proteins. Under oxidizing conditions, the mutants produced higher 1-anilinonaphthalene-8-sulfonic acid and thioflavin T fluorescence signals than the WT, indicating increased protein unfolding and fibril formation, respectively. Finally, far-ultraviolet circular dichroism spectroscopy revealed that WT TGFBIp undergoes concentration-dependent conformational changes; similar experiments were not possible on mutant TGFBIp, which remained soluble only at low concentrations. Conclusions: Our study provides new evidence for the pathogenic mechanism of dystrophic mutants. Although mutant TGFBIp has moderate but consistent structural perturbations, other factors such as oxidation or degradation may be required to cause the phenotypic abnormal aggregations.